Nucleases, including zinc finger nucleases and homing endonucleases such as I-SceI, that are engineered to specifically bind to target sites have been shown to be useful in genome engineering in basic research and in the pharmaceutical and biotechnology applications. For example, zinc finger nucleases (ZFNs) are proteins comprising engineered site-specific zinc fingers fused to a nuclease domain. Such ZFNs have been successfully used for genome modification in a variety of different species. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. These ZFNs can be used to create a double-strand break (DSB) in a target nucleotide sequence, which increases the frequency of homologous recombination at the targeted locus (targeted integration) more than 1000-fold. In addition, the inaccurate repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene disruption. Creation of two such DSBs results in deletion of arbitrarily large regions.
As nuclease-mediated genome modification facilitates basic science research and development of therapeutics, in vitro and in vivo assays have been developed to measure the activity of ZFNs. See, e.g., WO 2009/042163. These assays are based on different pathways to repair DNA double-strand breaks (DSBs) catalyzed by the recruitment of ZFNs to a pre-determined location in the genome of eukaryotic cells. DSB are repaired by either non-conservative non-homologous end joining (NHEJ) pathways or the conservative homology directed repair (HDR). In addition, a non-conservative HDR pathway called single-strand-annealing (SSA) is also present in most cells. The SSA pathway shares some of the cellular machinery with the HR pathway.
In addition to detecting biologically active nucleases by measuring their increased capacity to bind and cleave their intended loci in the genome, it is also desirable to identify and enrich for cells having the desired nuclease-mediated genomic modifications. Currently, many existing methods rely on the integration and expression of a drug selection marker into the desired locus. The marker and/or drug selection genes are often integrated into the cell genome permanently or exist in an episomal form for a long period of time. The presence of these genetic elements in the final cell clone is often undesirable. Also, the high incidence of random integration can create a high background of cell clones with no modification at the intended target locus.
Thus, there remains a need for additional assays to screen for nuclease activity and to identify cells with the desired genomic modifications without using drug selection or a permanent marker that is integrated into the genome.